RESUMEN

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Asunto(s)

Plaquetas , Factor de Transcripción GATA1 , Megacariocitos , Trombopoyesis , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Humanos , Animales , Plaquetas/metabolismo , Trombopoyesis/genética , Megacariocitos/metabolismo , Megacariocitos/citología , Mutación , Trombocitopenia/genética , Trombocitopenia/patología , Trombocitopenia/metabolismo , Diferenciación Celular/genética , Ratones

RESUMEN

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-ß (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-ß) signaling and TGF-ß trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-ß content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-ß or CXCL1).

Asunto(s)

Janus Quinasa 1 , Selectina-P , Mielofibrosis Primaria , Pirimidinas , Receptores de Interleucina-8B , Factor de Crecimiento Transformador beta , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Selectina-P/metabolismo , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8A/metabolismo , Ratones , Janus Quinasa 2/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Nitrilos/uso terapéutico , Nitrilos/farmacología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/genética , Pirazoles/farmacología , Pirazoles/uso terapéutico , Humanos

RESUMEN

Transcriptional mechanisms establish and maintain complex genetic and protein networks to control cell state transitions. The hematopoietic transcription factor GATA1 is a master regulator of erythropoiesis and megakaryopoiesis, and human GATA1 genetic variants cause anemia and megakaryoblastic leukemia. Multiomic analyses revealed that GATA1 controls expression of transporters and metabolic enzymes that dictate intracellular levels of endogenous small molecules, including heme, metal ions, and sphingolipids. Besides its canonical function as a hemoglobin component, heme facilitates or antagonizes GATA1 function to regulate erythropoiesis via mechanisms dependent or independent of the heme-binding transcription factor BTB domain and CNC homology 1 (BACH1). GATA1 regulates the expression of genes encoding heme biosynthetic enzymes and BACH1. GATA1 maintains homeostasis of bioactive ceramides during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Disrupting ceramide homeostasis impairs critical cytokine signaling and is detrimental to erythroid cells. During erythroid maturation, GATA1 induces a zinc transporter switch that favors export versus import, thus dictating the intracellular zinc level, erythroblast survival, and differentiation. In aggregate, these studies support an emerging paradigm in which GATA factor-dependent transcriptional mechanisms control the intracellular levels of endogenous small molecules and small molecule-dependent feedback loops that serve as vital effectors of transcription factor activity, genome function, and cell state transitions.

Asunto(s)

Eritropoyesis , Humanos , Eritropoyesis/genética , Animales , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/genética , Factores de Transcripción GATA/metabolismo , Factores de Transcripción GATA/genética , Regulación de la Expresión Génica , Hemo/metabolismo , Hemo/biosíntesis , Diferenciación Celular

RESUMEN

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome is a distinct form of leukemia or preleukemia that mirrors the hematological features of acute megakaryoblastic leukemia. However, it typically resolves spontaneously in the early stages. TAM originates from fetal liver (FL) hematopoietic precursor cells and emerges due to somatic mutations in GATA1 in utero. In TAM, progenitor cells proliferate and differentiate into mature megakaryocytes and granulocytes. This process occurs both in vitro, aided by hematopoietic growth factors (HGFs) produced in the FL, and in vivo, particularly in specific anatomical sites like the FL and blood vessels. The FL's hematopoietic microenvironment plays a crucial role in TAM's pathogenesis and may contribute to its spontaneous regression. This review presents an overview of current knowledge regarding the unique features of TAM in relation to the FL hematopoietic microenvironment, focusing on the functions of HGFs and the pathological features of TAM.

Asunto(s)

Síndrome de Down , Reacción Leucemoide , Hígado , Humanos , Síndrome de Down/complicaciones , Síndrome de Down/patología , Hígado/patología , Reacción Leucemoide/genética , Reacción Leucemoide/patología , Reacción Leucemoide/diagnóstico , Reacción Leucemoide/complicaciones , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/metabolismo , Feto , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Mielopoyesis

RESUMEN

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Asunto(s)

Eritroblastos , Eritropoyesis , Factor de Transcripción GATA1 , Hemo , Lipoproteínas , Macrófagos , Policitemia , Policitemia/metabolismo , Policitemia/genética , Policitemia/patología , Eritroblastos/metabolismo , Hemo/metabolismo , Humanos , Animales , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Ratones , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/genética , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Trombomodulina/metabolismo , Trombomodulina/genética , Ratones Noqueados , Ferroquelatasa/metabolismo , Ferroquelatasa/genética , Masculino , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Femenino

RESUMEN

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Asunto(s)

Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas , Factores de Transcripción , Humanos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/genética , Ratones , Hematopoyesis , Proteínas que Contienen Bromodominio

RESUMEN

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP­like cell line HEL western blotting, RT­qPCR, lentivirus­mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation­specific (ETS) transcription factor friend leukemia integration factor 1 (Fli­1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1­mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation­specific­related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA­mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Asunto(s)

Diferenciación Celular , Células Eritroides , Megacariocitos , Humanos , Diferenciación Celular/genética , Línea Celular , Células Eritroides/metabolismo , Células Eritroides/citología , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Megacariocitos/metabolismo , Megacariocitos/citología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Regulador Transcripcional ERG/metabolismo , Regulador Transcripcional ERG/genética

Asunto(s)

Factor de Transcripción GATA1 , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Humanos , Animales , Trastornos de Fallo de la Médula Ósea/metabolismo , Trastornos de Fallo de la Médula Ósea/patología , Enfermedades de la Médula Ósea/metabolismo , Enfermedades de la Médula Ósea/patología , Enfermedades de la Médula Ósea/genética

RESUMEN

Toll-interacting protein (Tollip) serves as a crucial inhibitory factor in the modulation of Toll-like receptor (TLR)-mediated innate immunological responses. The structure and function of Tollip have been well documented in mammals, yet the information in teleost remained limited. This work employed in vitro overexpression and RNA interference in vivo and in vitro to comprehensively examine the regulatory effects of AjTollip on NF-κB and MAPK signaling pathways. The levels of p65, c-Fos, c-Jun, IL-1, IL-6, and TNF-α were dramatically reduced following overexpression of AjTollip, whereas knocking down AjTollip in vivo and in vitro enhanced those genes' expression. Protein molecular docking simulations showed AjTollip interacts with AjTLR2, AjIRAK4a, and AjIRAK4b. A better understanding of the transcriptional regulation of AjTollip is crucial to elucidating the role of Tollip in fish antibacterial response. Herein, we cloned and characterized a 2.2 kb AjTollip gene promoter sequence. The transcription factors GATA1 and Sp1 were determined to be associated with the activation of AjTollip expression by using promoter truncation and targeted mutagenesis techniques. Collectively, our results indicate that AjTollip suppresses the NF-κB and MAPK signaling pathways, leading to the decreased expression of the downstream inflammatory factors, and GATA1 and Sp1 play a vital role in regulating AjTollip expression.

Asunto(s)

Anguilla , Proteínas de Peces , Factor de Transcripción GATA1 , FN-kappa B , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , FN-kappa B/metabolismo , FN-kappa B/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Anguilla/genética , Anguilla/inmunología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/química , Transducción de Señal

RESUMEN

Mutations in the master hematopoietic transcription factor GATA1 are often associated with functional defects in erythropoiesis and megakaryopoiesis. In this study, we identified a novel GATA1 germline mutation (c.1162delGG, p.Leu387Leufs*62) in a patient with congenital anemia and occasional thrombocytopenia. The C-terminal GATA1, a rarely studied mutational region, undergoes frameshifting translation as a consequence of this double-base deletion mutation. To investigate the specific function and pathogenic mechanism of this mutant, in vitro mutant models of stable re-expression cells were generated. The mutation was subsequently validated to cause diminished transcriptional activity of GATA1 and defective differentiation of erythroid and megakaryocytes. Using proximity labeling and mass spectrometry, we identified selective alterations in the proximal protein networks of the mutant, revealing decreased binding to a set of normal GATA1-interaction proteins, including the essential co-factor FOG1. Notably, our findings further demonstrated enhanced recruitment of the protein arginine methyltransferase PRMT6, which mediates histone modification at H3R2me2a and represses transcription activity. We also found an enhanced binding of this mutant GATA1/PRMT6 complex to the transcriptional regulatory elements of GATA1's target genes. Moreover, treatment of the PRMT6 inhibitor MS023 could partially rescue the inhibited transcriptional and impaired erythroid differentiation caused by the GATA1 mutation. Taken together, our results provide molecular insights into erythropoiesis in which mutation leads to partial loss of GATA1 function, and the role of PRMT6 and its inhibitor MS023 in congenital anemia, highlighting PRMT6 binding as a negative factor of GATA1 transcriptional activity in aberrant hematopoiesis.

Asunto(s)

Factor de Transcripción GATA1 , Mutación de Línea Germinal , Unión Proteica , Proteína-Arginina N-Metiltransferasas , Humanos , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Diferenciación Celular/genética , Eritropoyesis/genética , Masculino , Femenino , Anemia/genética

RESUMEN

Rho GTPase activating protein 18 (ARHGAP18), a member of the RhoGAP gene family that increases GTP hydrolysis and inhibits RhoGTPase, was recently discovered to play a role in the development of breast cancer. However, its exact biological role in hepatocellular carcinoma (HCC) remains unclear. In our present study, we comprehensively assessed ARHGAP18 expression and its correlation with the prognostic value of cancer patients in databases. Cell proliferation and colony formation assays were employed to monitor cell growth. Luciferase reporter assay, Chromatin immunoprecipitation qPCR (ChIP-qPCR), immunofluorescence were performed for mechanism research. The expression of genes and proteins was detected by real-time PCR and western blotting. According to the findings of this research, ARHGAP18 protein levels are increased in HCC tissues compared to adjacent nontumor tissues, and ARHGAP18 overexpression is associated with poor survival. The results of a gain- and loss-of-function experiment with HCC cells in vitro demonstrated that ARHGAP18 stimulated cell proliferation, migration, and invasion. Mechanistically, we found that the transcription factor GATA binding protein 1 (GATA1) could bind to the ARHGAP18 promoter and facilitate ARHGAP18 expression. Further studies revealed that the effects of ARHGAP18 silencing on HCCLM3 and Bel-7402 cells were blocked by GATA1 overexpression. In conclusion, GATA1-mediated ARHGAP18 up-regulation plays an important role in HCC tumorigenesis and might be a potential therapeutic target for HCC.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo

RESUMEN

BACKGROUND: The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES: To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS: We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS: We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 µm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION: Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Asunto(s)

Anemia , Factor de Transcripción GATA1 , Miosina Tipo IIA no Muscular , Proteínas Proto-Oncogénicas , Proteínas Represoras , Trombocitopenia , Humanos , Plaquetas/metabolismo , Eritrocitos , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética

RESUMEN

INTRODUCTION: GATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. OBJECTIVE: To determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. METHODS: Comprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. RESULTS: The patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbß3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. CONCLUSIONS: The G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

Asunto(s)

Trastornos de las Plaquetas Sanguíneas , Trombocitopenia , Humanos , Trombina/metabolismo , Calcio/metabolismo , Plaquetas/metabolismo , Trombocitopenia/patología , Activación Plaquetaria , Factor de Transcripción GATA1/metabolismo

RESUMEN

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

Asunto(s)

Factor de Transcripción GATA1 , Factores de Transcripción , Ratones , Humanos , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Regiones Promotoras Genéticas/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo

RESUMEN

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

Asunto(s)

Citocinas , Redes Reguladoras de Genes , Citocinas/genética , Proteómica , Factor de Transcripción GATA1/metabolismo , Diferenciación Celular/genética , Ceramidas , Homeostasis

RESUMEN

ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.

Asunto(s)

Eritropoyesis , Factores de Transcripción , Humanos , Eritropoyesis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Globinas beta/genética , Globinas beta/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Genetic determinants underlying most human blood groups are now clarified but variation in expression levels remains largely unexplored. By developing a bioinformatics pipeline analyzing GATA1/Chromatin immunoprecipitation followed by sequencing (ChIP-seq) datasets, we identify 193 potential regulatory sites in 33 blood-group genes. As proof-of-concept, we aimed to delineate the low-expressing complement receptor 1 (CR1) Helgeson phenotype on erythrocytes, which is correlated with several diseases and protects against severe malaria. We demonstrate that two candidate CR1 enhancer motifs in intron 4 bind GATA1 and drive transcription. Both are functionally abolished by naturally-occurring SNVs. Erythrocyte CR1-mRNA and CR1 levels correlate dose-dependently with genotype of one SNV (rs11117991) in two healthy donor cohorts. Haplotype analysis of rs11117991 with previously proposed markers for Helgeson shows high linkage disequilibrium in Europeans but explains the poor prediction reported for Africans. These data resolve the longstanding debate on the genetic basis of inherited low CR1 and form a systematic starting point to investigate the blood group regulome.

Asunto(s)

Células Eritroides , Factor de Transcripción GATA1 , Receptores de Complemento 3b , Humanos , Pueblo Africano , Biología Computacional , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Genotipo , Intrones , Fenotipo , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Células Eritroides/metabolismo , Pueblo Europeo

RESUMEN

Mammalian erythroid development can be divided into three stages: hematopoietic stem and progenitor cell (HSPC), erythroid progenitor (Ery-Pro), and erythroid precursor (Ery-Pre). However, the mechanisms by which the 3D genome changes to establish the stage-specific transcription programs that are critical for erythropoiesis remain unclear. Here, we analyze the chromatin landscape at multiple levels in defined populations from primary human erythroid culture. While compartments and topologically associating domains remain largely unchanged, ∼50% of H3K27Ac-marked enhancers are dynamic in HSPC versus Ery-Pre. The enhancer anchors of enhancer-promoter loops are enriched for occupancy of respective stage-specific transcription factors (TFs), indicating these TFs orchestrate the enhancer connectome rewiring. The master TF of erythropoiesis, GATA1, is found to occupy most erythroid gene promoters at the Ery-Pro stage, and mediate conspicuous local rewiring through acquiring binding at the distal regions in Ery-Pre, promoting productive erythroid transcription output. Knocking out GATA1 binding sites precisely abrogates local rewiring and corresponding gene expression. Interestingly, knocking down GATA1 can transiently revert the cell state to an earlier stage and prolong the window of progenitor state. This study reveals mechanistic insights underlying chromatin rearrangements during development by integrating multidimensional chromatin landscape analyses to associate with transcription output and cellular states.

Asunto(s)

Cromatina , Eritropoyesis , Factor de Transcripción GATA1 , Animales , Humanos , Diferenciación Celular , Cromatina/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética

RESUMEN

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

Asunto(s)

Anemia de Diamond-Blackfan , Proteínas de Unión a la Región de Fijación a la Matriz , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Megacariocitos/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Diferenciación Celular/genética , Factores de Transcripción/metabolismo , Anemia de Diamond-Blackfan/metabolismo , Cromatina/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo

RESUMEN

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Eritropoyesis , Factor de Transcripción GATA1 , Proteínas Represoras , Animales , Humanos , Ratones , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Eritroides/metabolismo , Factor de Transcripción GATA1/metabolismo , Perfilación de la Expresión Génica , Policitemia Vera/genética , Policitemia Vera/metabolismo , Proteínas Represoras/metabolismo